Cardiomyocyte proliferation plays a crucial role in the developing mammal heart, as it is required for normal morphogenesis and in determining the appropriate heart size. Postnatally, the decrease in cell cycle activity is concomitant with the increase of cell cycle variants, such as endoreduplication and acytokinetic mitosis, which contribute to the hypertrophic growth of the heart. Although the adult mammal heart retains the ability to generate new cardiomyocytes, the extent of cardiomyocyte renewal is insufficient to compensate for the large-scale tissue loss associated with ischemic events. Indeed, ischemic events such as myocardial infarction, lead to a permanent loss of ventricular cardiomyocytes, formation of collagen-containing scar, and consequently cardiac remodeling. The development of therapies able to hamper cardiac remodeling by promoting cardiomyocyte turnover is one of the primary goals in the cardiovascular field. In the present study, we generated a human induced pluripotent stem cell (iPSC) line containing the fluorescence ubiquitination-based cell cycle indicator (FUCCI) under the Troponin T2 (TNNT2) promoter. To gain information about the cell cycle dynamics of human cardiomyocytes, we visualized cell cycle progression in TNNT2-FUCCI human iPSCs-derived cardiomyocytes. Notably, we revealed cardiomyocytes' cell cycle dynamics of cells undergoing proliferation, binucleation, and polyploidization and identified G2 cell cycle arrest in cardiomyocytes undergoing polyploidization. To demonstrate the versatility of the TNNT2-FUCCI human iPSCs line, we developed a live cell screening platform to identify pro-proliferative compounds within an autophagy compound library. We identified Clonidine, an alpha2-adrenergic receptor and imidazoline agonist, as an enhancer of cell cycle activity in TNNT2-FUCCI hiPSC-derived cardiomyocytes. Finally, we investigated the ability of Clonidine to promote cell cycle progression in hiPSC- derived cardiomyocytes and in in vivo and in vitro mouse neonatal cardiomyocytes. We showed that while Clonidine stimulated cardiomyocytes' polyploidization and multinucleation, respectively in in vitro in in vivo mouse cardiomyocytes, the treatment of hiPSC-derived cardiomyocytes with Clonidine enhanced their proliferative capability. In conclusion, we showed that the TNNT2-FUCCI system is a versatile tool for characterizing cardiomyocyte cell cycle dynamics and identifying pro-proliferative molecular candidates with regenerative potential in the mammalian heart.:1. Introduction 1.1 Heart function and composition 1.2 Human cardiac development 1.2.1 Cardiac organogenesis 1.2.2 Metabolic changes in the developing heart 1.3 Cardiomyocytes cell cycle activity 1.3.1 Cardiomyocytes cell cycle regulators and cell cycle arrest 1.3.2 CM multinucleation and polyploidization 1.4. The regenerative capabilities of the mammal heart 1.4.1 Model systems for heart regeneration 1.4.2 Stimulation of cardiomyocyte proliferation as a goal to preserve heart function 1.4.3 Assessment of cardiomyocytes proliferation 1.5 Human-induced pluripotent stem cells to model cardiac development and disease 2. Aim 3. Materials and methods 3.1 TNNT2-FUCCI hiPSC line generation 3.2 hiPSC culture and maintenance 3.3 hiPSC differentiation into CMs 3.4 Imagestream-X Analysis 3.5 TNNT2 expression assessment of hiPSC-derived CMs by immunohistochemistry 3.6 CDK1 immunohistochemistry expression assessment of hiPSC-derived CMs 3.7 Cell area and sarcomere spacing measurement of hiPSC-derived CMs 3.8 Live imaging and timelapse imaging analysis of TNNT2-FUCCI hiPSC 3.9 Murine neonatal CMs cell culture 3.10 Mouse nCM timelapse imaging and analysis 3.11 TNNT2-FUCCI hiPSC-derived CMs culturing and screen conditions 3.11.1 TNNT2-FUCCI screen image acquisition 3.11.2 TNNT2-FUCCI screen automated image analysis 3.11.3 TNNT2-FUCCI screen data analysis 3.12 Primary mouse nCM compound validation and immunohistochemistry 3.13 Mouse nCM immunohistochemistry for AurKB expression assessment 3.14 hiPSC-derived CMs immunohistochemistry for AurKB expression assessment 3.15 Analysis of CM ploidy and binucleation 3.16 in vivo Clonidine treatment of neonatal mice 3.17 Analysis of ploidy and binucleation in in vivo mouse nCMs 3.18 Cell cycle activity assessment in P7 neonatal mouse hearts after Clonidine treatment by immunohistochemistry 4. Results 4.1 TNNT2-FUCCI human iPSC line generation and validation 4.2 TNNT2-FUCCI marks proliferating and non-proliferating CMs 4.3 Live imaging identification of CM cell cycle activity 4.3.1 Cell cycle progression of TNNT2-FUCCI CMs 4.3.2 Cell cycle progression of mouse neonatal cardiomyocytes 4.4 TNNT2-FUCCI live-imaging identifies CM cell cycle activators 4.5 Compound validation in mouse nCMs 4.6 Clonidine elicits cycling activity via alpha1 adrenergic receptor and imidazoline receptor interaction 4.7 Clonidine stimulates hiPSC-derived CM proliferation 4.8 Clonidine stimulates CM polyploidization in mouse nCMs 4.9 Clonidine mediates in vivo CM cell cycle activity in the neonatal mouse 5. Discussion 5.1 TNNT2-FUCCI hiPSC: a new technology to identify cycling CMs 5.2 Study of CM cell cycle activity using TNNT2-FUCCI 5.3 TNNT2-FUCCI hiPSC in combination with a live screening platform revealed enhancer of CMs cell cycle activity. 5.4 Initial validation of pro-proliferative compounds in mouse nCMs reveals Clonidine and Dihydrocapsaicin as enhancers of cell cycle progression 5.5 Clonidine stimulates cell cycle activity in different model systems 5.5.1 Clonidine treatment stimulates proliferation in hiPSC-derived CMs 5.5.2 Clonidine treatment stimulates polyploidization in in vitro- and multinucleation in in vivo mouse nCMs 5.6 Conclusions and future outlooks 6. Appendix 7. Summary 8. Zusammenfassung Acknowledgements References Anlage 1 Anlage 2